Genetic consequences of a century of protection: serial founder events and survival of the little spotted kiwi (Apteryx owenii).

We present the outcome of a century of post-bottleneck isolation of a long-lived species, the little spotted kiwi (Apteryx owenii, LSK) and demonstrate that profound genetic consequences can result from protecting few individuals in isolation. LSK were saved from extinction by translocation of five birds from South Island, New Zealand to Kapiti Island 100 years ago. The Kapiti population now numbers some 1200 birds and provides founders for new populations. We used 15 microsatellite loci to compare genetic variation among Kapiti LSK and the populations of Red Mercury, Tiritiri Matangi and Long Islands that were founded with birds from Kapiti. Two LSK native to D'Urville Island were also placed on Long Island. We found extremely low genetic variation and signatures of acute and recent genetic bottleneck effects in all four populations, indicating that LSK have survived multiple genetic bottlenecks. The Long Island population appears to have arisen from a single mating pair from Kapiti, suggesting there is no genetic contribution from D'Urville birds among extant LSK. The Ne/NC ratio of Kapiti Island LSK (0.03) is exceptionally low for terrestrial vertebrates and suggests that genetic diversity might still be eroding in this population, despite its large census size.

Phylogeography of the endangered Otago skink, Oligosoma otagense: population structure, hybridisation and genetic diversity in captive populations.

Climatic cooling and substantial tectonic activity since the late Miocene have had a pronounced influence on the evolutionary history of the fauna of New Zealand's South Island. However, many species have recently experienced dramatic range reductions due to habitat fragmentation and the introduction of mammalian predators and competitors. These anthropogenic impacts have been particularly severe in the tussock grasslands of the Otago region. The Otago skink (Oligosoma otagense), endemic to the region, is one of the most critically endangered vertebrates in New Zealand. We use mitochondrial DNA sequence data to investigate the evolutionary history of the Otago skink, examine its population genetic structure, and assess the level of genetic diversity in the individuals in the captive breeding program. Our data indicate that the Otago skink diverged from its closest relatives in the Miocene, consistent with the commencement of tectonic uplift of the Southern Alps. However, there is evidence for past introgression with the scree skink (O. waimatense) in the northern Otago-southern Canterbury region. The remnant populations in eastern Otago and western Otago are estimated to have diverged in the mid-Pliocene, with no haplotypes shared between these two regions. This divergence accounts for 95% of the genetic diversity in the species. Within both regions there is strong genetic structure among populations, although shared haplotypes are generally evident between adjacent localities. Although substantial genetic diversity is present in the captive population, all individuals originate from the eastern region and the majority had haplotypes that were not evident in the intensively managed populations at Macraes Flat. Our data indicate that eastern and western populations should continue to be regarded as separate management units. Knowledge of the genetic diversity of the breeding stock will act to inform the captive management of the Otago skink and contribute to a key recovery action for the species.

New Zealand geckos (Diplodactylidae): Cryptic diversity in a post-Gondwanan lineage with trans-Tasman affinities.

We used a multi-gene approach to assess the phylogenetic relationships of New Zealand diplodactylid geckos to their Australian and New Caledonian relatives and to one another. Data from nuclear (RAG-1, PDC) and mitochondrial (ND2, 16S) genes from >180 specimens representing all 19 recognized New Zealand taxa and all but two of 20 putatively new species suggested by previous studies were analyzed using Maximum Parsimony, Maximum Likelihood and Bayesian inference. All analyses retrieved a monophyletic New Zealand clade, most closely related to the Australian Diplodactylidae exclusive of Pseudothecadactylus. Hoplodactylus is paraphyletic and composed of two morphological groups: a broad-toed clade, consisting of the island-restricted, largest extant species, Hoplodactylus duvaucelii, and the species-rich, wide-ranging Hoplodactylus maculatus clade; and a narrow-toed clade, comprising five monophyletic subgroups: Naultinus, the Hoplodactylus pacificus and Hoplodactylus granulatus clades, and the distinctive species Hoplodactylus rakiurae and Hoplodactylus stephensi. Each of these lineages is here recognized at the generic level. Our data support recognition of 16 new species (36 total), and five new or resurrected genera (seven total). The New Zealand diplodactylid radiation split from its Australian relatives 40.2mya (95% highest posterior density estimate 28.9-53.5), after the opening of the Tasman Sea. Their distribution cannot, therefore, be regarded as derived as a result of Gondwanana vicariance. The age of the New Zealand crown group, 24.4mya (95% highest posterior density estimate 15.5-33.8), encompasses the period of the 'Oligocene drowning' of New Zealand and is consistent with the hypothesis that New Zealand was not completely inundated during this period. Major lineages within New Zealand geckos diverged chiefly during the mid- to late Miocene, probably in association with a suite of geological and climatological factors that have characterized the region's complex history.

Genetic diversity and differentiation at MHC genes in island populations of tuatara (Sphenodon spp.).

Neutral genetic markers are commonly used to understand the effects of fragmentation and population bottlenecks on genetic variation in threatened species. Although neutral markers are useful for inferring population history, the analysis of functional genes is required to determine the significance of any observed geographical differences in variation. The genes of the major histocompatibility complex (MHC) are well-known examples of genes of adaptive significance and are particularly relevant to conservation because of their role in pathogen resistance. In this study, we survey diversity at MHC class I loci across a range of tuatara populations. We compare the levels of MHC variation with that observed at neutral microsatellite markers to determine the relative roles of balancing selection, diversifying selection and genetic drift in shaping patterns of MHC variation in isolated populations. In general, levels of MHC variation within tuatara populations are concordant with microsatellite variation. Tuatara populations are highly differentiated at MHC genes, particularly between the northern and Cook Strait regions, and a trend towards diversifying selection across populations was observed. However, overall our results indicate that population bottlenecks and isolation have a larger influence on patterns of MHC variation in tuatara populations than selection.

Nocturnal lizards from a cool-temperate environment have high metabolic rates at low temperatures.

Ectotherms from low-temperature environments have higher metabolic rates at low temperatures than those from warm-temperature environments. We predicted that nocturnal lizards, which are active at much lower environmental temperatures than diurnal lizards, would also have higher metabolic rates at low temperatures, and by association a lower thermal sensitivity (Q(10)) than diurnal and crepuscular lizards. We measured the rate of oxygen consumption (VO2) of eight cool-temperate species of lizard (four nocturnal, three diurnal, and one crepuscular) at 13 and 26°C and analyzed log transformations of these data using log mass as a covariate. As expected, VO2 was positively correlated with temperature in all eight species, with VO2 being two to four times higher at 26°C than at 13°C. As predicted, at 13°C (but not 26°C) the VO2 was significantly higher in nocturnal than diurnal lizards. Species-specific differences and mass scaling factors explain the patterns of thermal sensitivity seen among these eight lizard species. Thermal sensitivity is strongly influenced by mass, with smaller species generally having higher thermal sensitivity of their metabolic rate, and this result deserves further exploration among other ectotherms. We conclude that, along with the previously reported lower cost of locomotion found in nocturnal lizards, they also partially offset the thermal handicap of activity at low body temperatures by having an elevated VO2 at lower temperatures.

Origin, diversification, and systematics of the New Zealand skink fauna (Reptilia: Scincidae).

The diverse scincid lizard fauna of the largely submerged subcontinent of Zealandia (which incorporates New Zealand, New Caledonia, Lord Howe Island, Norfolk Island, and the Chatham Islands) forms a monophyletic lineage within the Eugongylus group of skinks. We use 4062 bp of mitochondrial (ND2, ND4, Cytochrome b, 12SrRNA, 16SrRNA) and nuclear (Rag-1) DNA sequence data to recover a molecular phylogeny for the New Zealand skink fauna, and investigate the origin and diversification of skinks in New Zealand. Our phylogeny includes 32 of the 33 extant described New Zealand skink species (Cyclodina and Oligosoma), the Lord Howe Island skink (C. lichenigera), and representatives from several New Caledonian genera. Neighbour-joining, Maximum Parsimony, Maximum Likelihood, and Bayesian phylogenetic analyses are used to demonstrate that the New Zealand skink species form a single monophyletic lineage, with C. lichenigera representing a closely related sister lineage to the New Zealand radiation. Our relaxed molecular clock analyses indicate that skinks colonised New Zealand in the early Miocene (16-22.6 mya), shortly after the 'Oligocene drowning' event (approximately 25 mya). We propose that skinks reached New Zealand from New Caledonia via long-distance overwater dispersal, with C. lichenigera persisting on volcanic islands along the Lord Howe Rise and Norfolk Ridge. Eight major genetic clades are evident within the New Zealand skink fauna, with the divergences among these clades during the early to mid-Miocene resulting in distinct open habitat, forest, and coastal radiations. Subsequent diversification in the late Miocene-Pliocene appears to coincide with tectonic activity along the Alpine Fault and the uplift of the Southern Alps. We were unable to resolve the phylogenetic affinities of O. suteri, New Zealand's only native oviparous skink. We use the phylogeny and topology tests to resolve several taxonomic issues and assess the taxonomic status of several suspected undescribed taxa. We complete a generic revision for the New Zealand skink fauna, placing C. lichenigera and all native New Zealand species into a single genus.

Influence of major histocompatibility complex genotype on mating success in a free-ranging reptile population.

Major histocompatibility complex (MHC) genes are highly polymorphic components of the vertebrate immune system, which play a key role in pathogen resistance. MHC genes may also function as odour-related cues for mate choice, thus ensuring optimal MHC diversity in offspring. MHC-associated mate choice has been demonstrated in some fish, bird and mammal species but it is not known whether this is a general vertebrate phenomenon. We investigated whether MHC-associated mate choice occurs in a wild population of tuatara (Sphenodon punctatus), a territorial and sexually dimorphic reptile. We found weak evidence for MHC-disassortative mating, based on amino acid genotypic distance between pairs, when mated pairs were directly compared with potential pairs in close spatial proximity. No significant association was found between male mating success, number of MHC sequences, microsatellite heterozygosity or MHC lineage. The major determinant of mating success in tuatara was male body size, which was not related to MHC lineage or microsatellite heterozygosity. Our results suggest that male competitive ability is the primary driver of mating success in tuatara. However, MHC-associated preferences also appear to play a role, possibly as a kin avoidance mechanism during territory formation.

Discrimination of flicker frequency rates in the reptile tuatara (Sphenodon ).

By investigating the mechanisms that underlie the perception of environmental cues, we may begin to understand how the sensory system governs behavioral responses. This is the first empirical study to examine learning and visual sensitivity in a reptile species, the tuatara (Sphenodon punctatus). We established a non-intrusive psychophysical method by employing an instrumental paradigm in order to examine discrimination learning and the ability to distinguish different flicker frequencies in the tuatara. Seventeen tuatara were trained under an operant conditioning task to respond to various discriminative stimuli flickering between 2.65 and 65.09 Hz. Tuatara were able to learn the operant task and discriminate between a constant light and flicker frequency rates between 2.65 and 45.61 Hz, but not at 65.09 Hz. We demonstrated a reliable psychophysical method where these reptiles could learn a basic operant task and discriminate visual stimuli in the form of flicker frequency rates. The tuatara's ability to perceive flickering light is comparable to that of avian, mammalian, and other reptilian species. This method is thus suitable for more comprehensive examinations of vision and additional sensory abilities in other reptiles.

A SINE of restricted gene flow across the Alpine Fault: phylogeography of the New Zealand common skink (Oligosoma nigriplantare polychroma).

New Zealand has experienced a complex climatic and geological history since the Pliocene. Thus, identifying the processes most important in having driven the evolution of New Zealand's biota has proven difficult. Here we examine the phylogeography of the New Zealand common skink (Oligosoma nigriplantare polychroma) which is distributed throughout much of New Zealand and crosses many putative biogeographical boundaries. Using mitochondrial DNA sequence data, we revealed five geographically distinct lineages that are highly differentiated (pairwise Phi(ST) 0.54-0.80). The phylogeographical pattern and inferred age of the lineages suggests Pliocene mountain building along active fault lines promoted their divergence 3.98-5.45 million years ago. A short interspersed nuclear element (SINE) polymorphism in the myosin gene intron (MYH-2) confirmed a pattern of restricted gene flow between lineages on either side of the mountain ranges associated with the Alpine Fault that runs southwest to northeast across the South Island of New Zealand. An analysis of molecular variance confirmed that approximately 40% of the genetic differentiation in O. n. polychroma is distributed across this major fault line. The straits between the main islands of New Zealand accounted for much less of the variation found within O. n. polychroma, most likely due to the repeated existence of landbridges between islands during periods of the Pleistocene that allowed migration. Overall, our findings reveal the relative roles of different climatic and geological processes, and in particular, demonstrate the importance of the Alpine Fault in the evolution of New Zealand's biota.

Phylogeography of two New Zealand lizards: McCann's skink (Oligosoma maccanni) and the brown skink (O. zelandicum).

The New Zealand skink fauna has proven to be an ideal taxonomic group in which to examine the impact of climatic and geological processes on the evolution of the New Zealand biota since the Pliocene. Here we examine the phylogeography of McCann's skink (Oligosoma maccanni) in order to gain insight into the relative contribution of Pliocene and Pleistocene processes on patterns of genetic structure in the South Island biota, and investigate the phylogeography of the brown skink (O. zelandicum) to examine whether Cook Strait landbridges facilitated geneflow between the North and South Islands in the late-Pleistocene. We obtained mitochondrial DNA sequence data (ND2 and ND4; 1282bp) from across the range of both species. We examined the phylogeographic patterns evident in each species using Neighbour-Joining, Maximum Likelihood and Bayesian methods. We found substantial phylogeographic structure within O. maccanni, with seven distinct clades identified. Divergences among clades are estimated to have occurred during the Pliocene. Populations in the Otago/Southland region (south of the Waitaki River valley) formed a well-supported lineage within O. maccanni. A substantial genetic break was evident between populations in east and west Otago, either side of the Nevis-Cardrona fault system, while north-south genetic breaks were evident within the Canterbury region. Within-clade divergences in O. maccanni appear to have occurred during the mid- to late-Pleistocene. Shimodaira-Hasegawa topology tests indicated that the 'Garston' skink is not genetically distinct from O. maccanni. There was only relatively minor phylogeographic structure within O. zelandicum, with divergences among populations occurring during the mid- to late-Pleistocene. Our genetic data supports a single colonisation of the North Island by O. zelandicum from the South Island, with the estimated timing of this event (0.46mya) consistent with the initial formation of Cook Strait.

Origin and post-colonization evolution of the Chatham Islands skink (Oligosoma nigriplantare nigriplantare).

Island ecosystems provide an opportunity to examine a range of evolutionary and ecological processes. The Chatham Islands are an isolated archipelago situated approximately 800 km east of New Zealand. Geological evidence indicates that the Chatham Islands re-emerged within the last 1-4 million years, following a prolonged period of marine inundation, and therefore the resident flora and fauna is the result of long-distance overwater dispersal. We examine the origin and post-colonization evolution of the Chatham Islands skink, Oligosoma nigriplantare nigriplantare, the sole reptile species occurring on the archipelago. We sampled O. n. nigriplantare from across nine islands within the Chatham Islands group, and representative samples from across the range of its closest relative, the New Zealand mainland common skink (Oligosoma nigriplantare polychroma). Our mitochondrial sequence data indicate that O. n. nigriplantare diverged from O. n. polychroma 5.86-7.29 million years ago. This pre-dates the emergence date for the Chatham Islands, but indicates that O. n. nigriplantare colonized the Chatham Islands via overwater dispersal on a single occasion. Despite the substantial morphological variability evident in O. n. nigriplantare, only relatively shallow genetic divergences (maximum divergence approximately 2%) were found across the Chatham Islands. Our analyses (haplotypic diversity, Phi(ST), analysis of molecular variance, and nested clade phylogeographical analysis) indicated restricted gene flow in O. n. nigriplantare resulting in strong differentiation between islands. However, the restrictions to gene flow might have only arisen recently as there was also a significant pattern of isolation by distance, possibly from when the Chatham Islands were a single landmass during Pleistocene glacial maxima when sea levels were lower. The level of genetic and morphological divergence between O. n. nigriplantare and O. n. polychroma might warrant their recognition as distinct species.

Comparative phylogeography of three skink species (Oligosoma moco, O. smithi, O. suteri; Reptilia: Scincidae) in northeastern New Zealand.

Sea-level fluctuations during the Pliocene and Pleistocene have shaped the landscape of the Northland region of New Zealand. We examined the comparative phylogeography of three skink species (Oligosoma moco, O. smithi, O. suteri) in northeastern New Zealand in order to investigate the impact of the historical processes that have prevailed since the Pliocene on the Northland fauna. O. moco, O. smithi and O. suteri have similar distributions across northeastern New Zealand, frequently occurring in sympatry. We obtained sequence data from across the entire range of each species, targeting the ND2 mitochondrial gene. Using Neighbor-Joining, Maximum likelihood and Bayesian methods, our analysis revealed contrasting phylogeographic patterns in each species. We found substantial phylogeographic structure within O. moco, with three distinct clades identified. Similarly, deep phylogeographic divergence was evident within O. smithi, with three distinct clades present. Clade 1 included O. smithi populations from the Three Kings Islands and the western coastline of Northland, while Clade 2 encompassed the remainder of the range. However, since Clade 3 corresponded to a described species (O. microlepis), O. smithi might represent a species complex. In both O. moco and O. smithi, divergences among clades are estimated to have occurred in the Pliocene, with divergences within clades occurring during the Pleistocene. In contrast, genetic divergence among O. suteri populations was extremely limited and indicative of more recent divergences during the Pleistocene. The lack of phylogeographic structure in O. suteri might be a consequence of its oviparous reproductive mode, which restricted its distribution to warm northern refugia during glacial maxima. Differences in the ecology and biology of each species might have produced contrasting responses to the same historical processes, and ultimately diverse phylogeographic patterns. Our study reveals an absence of consistent and concordant phylogeographic patterns in the Northland biota, even within the same taxonomic group.

Two patterns of variation among MHC class I loci in Tuatara (Sphenodon punctatus).

The genes of the major histocompatibility complex (MHC) are a central component of the immune system in vertebrates and have become important markers of functional, fitness-related genetic variation. We have investigated the evolutionary processes that generate diversity at MHC class I genes in a large population of an archaic reptile species, the tuatara (Sphenodon punctatus), found on Stephens Island, Cook Strait, New Zealand. We identified at least 2 highly polymorphic (UA type) loci and one locus (UZ) exhibiting low polymorphism. The UZ locus is characterized by low nucleotide diversity and weak balancing selection and may be either a nonclassical class I gene or a pseudogene. In contrast, the UA-type alleles have high nucleotide diversity and show evidence of balancing selection at putative peptide-binding sites. Twenty-one different UA-type genotypes were identified among 26 individuals, suggesting that the Stephens Island population has high levels of MHC class I variation. UA-type allelic diversity is generated by a mixture of point mutation and gene conversion. As has been found in birds and fish, gene conversion obscures the genealogical relationships among alleles and prevents the assignment of alleles to loci. Our results suggest that the molecular mechanisms that underpin MHC evolution in nonmammals make locus-specific amplification impossible in some species.

Support for a rare pattern of temperature-dependent sex determination in archaic reptiles: evidence from two species of tuatara (Sphenodon).

BACKGROUND: The sex of many reptiles is determined by the temperature an embryo experiences during its development. Three patterns of temperature-dependent sex determination (TSD) have been defined, but one pattern where only males are produced above an upper temperature threshold (Type IB) is controversial. Here we report new data on the relationship between constant temperature incubation and sexual phenotype in two species of tuatara (Sphenodon), archaic reptiles of enormous zoological significance as the sole representatives of a once widespread reptilian order. RESULTS: In both species, the pattern observed with constant incubation temperatures from 18 to 23 degrees C (or 24 degrees C) supported a female-->male (FM or Type IB) pattern of TSD: in Sphenodon guntheri males were produced above a pivotal temperature of 21.6 degrees C, and in S. punctatus (unnamed subspecies on Stephens Island, Cook Strait), males were produced above a pivotal temperature of 22.0 degrees C. The pivotal temperatures and scaling parameters differed between species (p < 0.001). The thermosensitive period (TSP), where temperature influences gonad morphogenesis, occurs between 0.25 and 0.55 of embryonic development. While it is possible that the more common female-->male-->female (FMF or Type II) pattern exists, with a second pivotal temperature above 23-24 degrees C, we review several lines of evidence to the contrary. Most notably, we show that in S. punctatus, the warmest natural nests during the TSP produce predominantly males. CONCLUSION: An FM pattern of TSD could be currently adaptive in promoting sexual size dimorphism in tuatara. However, an FM pattern has particularly serious consequences for S. guntheri because current patterns of global warming could exacerbate the male bias already present in the relic population.

Daily patterns of metabolic rate among New Zealand lizards (Reptilia: Lacertilia: Diplodactylidae and Scincidae).

In addition to the effects of temperature fluctuations on metabolic rate, entrained endogenous rhythms in metabolism, which are independent of temperature fluctuations, may be important in overall energy metabolism in ectotherms. Daily entrained endogenous rhythms may serve as energy-conserving mechanisms during an animal's active or inactive phase. However, because nocturnal lizards often take advantage of thermal opportunities during the photophase (light), their daily metabolic rhythms may be less pronounced than those of diurnal species. We measured the rate of oxygen consumption (VO(2)) as an index of metabolic rate of eight temperate lizard species (four nocturnal, three diurnal, and one crepuscular/diurnal; n = 7-14) over 24 h at 13 degrees C and in constant darkness to test whether daily patterns (including amplitude, magnitude, and time of peak VO(2)) of metabolic rate in lizards differ with activity period. We also tested for phylogenetic differences in metabolic rate between skinks and geckos. Three daily patterns were evident: 24-h cycle, 12-h cycle, or no daily cycle. The skink Cyclodina aenea has a 12-h crepuscular pattern of oxygen consumption. In four other species, VO(2) increased with, or in anticipation of, the active part of the day, but three species had rhythms offset from their active phase. Although not correlated with activity period or phylogeny, amplitude of VO(2) may be correlated with whether a species is temperate or tropical. In conclusion, the metabolic rate of many species does not always correlate with the recorded activity period. The dichotomy of ecology and physiology may be clarified by more in-depth studies of species behaviors and activity periods.

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. MHC Class I genes in the Tuatara (Sphenodon spp.): evolution of the MHC in an ancient reptilian order.

The major histocompatibility complex (MHC) is an extremely dynamic region of the genome, characterized by high polymorphism and frequent gene duplications and rearrangements. This has resulted in considerable differences in MHC organization and evolution among vertebrate lineages, particularly between birds and mammals. As nonavian reptiles are ancestral to both mammals and birds, they occupy an important phylogenetic position for understanding these differences. However, little is known about reptile MHC genes. To address this, we have characterized MHC class I sequences from the tuatara (Sphenodon spp.), the last survivor of an ancient order of reptiles, Sphenodontia. We isolated two different class I cDNA sequences, which share 93% sequence similarity with each other but are highly divergent from other vertebrate MHC genes. Southern blotting and polymerase chain reaction amplification of class I sequences from seven adult tuatara plus a family group indicate that these sequences represent at least two to three loci. Preliminary analysis of variation among individuals from an island population of tuatara indicates that these loci are highly polymorphic. Maximum likelihood analysis of reptile MHC class I sequences indicates that gene duplication has occurred within reptilian orders. However, the evolutionary relationships among sequences from different reptilian orders cannot be resolved, reflecting the antiquity of the major reptile lineages.

Characterization of MHC class II genes from an ancient reptile lineage, Sphenodon (tuatara).

The organization and evolution of major histocompatibility complex (MHC) genes vary considerably among vertebrate lineages. MHC genes have been well characterized in mammals, birds, amphibians and fish, but little is known about their organization in reptiles, despite the fact that reptiles occupy an important phylogenetic position for understanding the evolutionary history of both mammalian and avian MHC genes. Here we describe the characterization of the first MHC class II B cDNA sequences from a non-avian reptile, the tuatara (Sphenodon spp.). Three class II B sequences were isolated from a tuatara cDNA library, and four additional partial sequences were isolated by reverse transcriptase-polymerase chain reaction. Six of these sequences appear to belong to the same gene family, which we have named SppuDAB. The remaining sequence (named SppuDBB) shares only 43.9% amino acid similarity with SppuDAB and thus appears to represent a separate gene family. SppuDBB may be a non-classical locus as it does not contain all the conserved residues expected of a classical MHC class II gene. Southern blot analysis indicates that only a single copy of SppuDBB exists in tuatara, but that multiple loci related to SppuDAB are present. The SppuDAB sequences have the highest amino acid similarity (57.2-62.4%) with class II B sequences from the spectacled caiman, but only 26.4-48.7% similarity with sequences from other vertebrates. The tuatara sequences do not strongly group with other reptile sequences on a phylogenetic tree, reflecting the antiquity of the Sphenodon lineage and the lack of closely related sequences for comparison.

T cell function in tuatara (Sphenodon punctatus).

Tuatara are the sole survivors of an entire order of reptiles that thrived during the age of the dinosaurs. Therefore, knowledge of their physiology is critical to understanding the phylogeny of reptiles. Previous studies of the immune system of the tuatara did not assess T cell function. We analyzed T cell function among six captive tuatara by assessing concanavalin A (Con A), phytohemagglutinin (PHA) and mixed lymphocyte reaction (MLR) induced T cell proliferation. Peripheral blood mononuclear cells from six out of six and four out of four tuatara tested exhibited significant proliferative responses to Con A and PHA, respectively, as measured by an MTT reduction assay. A lower level of proliferation was detected in an MLR. However, Con A activated lymphocytes were not cytotoxic for a xenogeneic murine mastocytoma cell line (P815).